Elevated endosomal cholesterol levels in Niemann-Pick cells inhibit Rab4 and perturb membrane recycling
Elevated endosomal cholesterol levels in Niemann-Pick cells inhibit Rab4 and perturb membrane recycling
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DOI：
10.1091/mbc.e04-05-0432
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发表时间：
2004-10-01
影响因子：
3.3
通讯作者：
Pagano, RE
中科院分区：
文献类型：
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作者：
Choudhury, A;Sharma, DK;Pagano, RE
在正常人皮肤成纤维细胞（HSF）中，荧光鞘糖脂类似物被内吞并分选成两个池，一个被回收到质膜，一个被转运到高尔基复合体。在这里，我们研究了鞘糖脂循环尼曼-匹克A型和C型脂质沉积病成纤维细胞（NPF）。将细胞与乳糖神经酰胺（LacCer）的荧光类似物在16 ℃下孵育以标记早期内体（EE），转移至37 ℃，并定量脂质再循环。利用显性阴性rabs，我们发现，在正常HSF中，LacCer再循环是快速的（t（1/2）类似于8分钟），主要依赖于rab 4。在NPF中，LacCer再循环延迟（t（1/2）类似于30 -40 min），不存在rab 4依赖的再循环，而rab 11依赖的再循环占主导地位。通过rab 4途径的转铁蛋白再循环在NPF中同样受到干扰。与正常的HSF相比，EEs在NPF显示高胆固醇水平和改变组织的Rab 4。在体外提取的rab 4（但不是rab 11）与GDP解离抑制剂严重衰减NPF内体组分。这种损害被逆转与胆固醇消耗分离的内体或与高盐处理的内体。这些数据表明，异常的膜回收NPFs的结果从特定的抑制rab 4功能的过量胆固醇在EE。
In normal human skin fibroblasts (HSFs), fluorescent glycosphingolipid analogues are endocytosed and sorted into two pools, one that is recycled to the plasma membrane and one that is transported to the Golgi complex. Here, we investigated glycosphingolipid recycling in Niemann-Pick type A and C lipid storage disease fibroblasts (NPFs). Cells were incubated with a fluorescent analogue of lactosylceramide (LacCer) at 16degreesC to label early endosomes (EEs), shifted to 37degreesC, and lipid recycling was quantified. Using dominant negative rabs, we showed that, in normal HSFs, LacCer recycling was rapid (t(1/2) similar to8 min) and mainly rab4-dependent. In NPFs, LacCer recycling was delayed (t(1/2) similar to30-40 min), and rab4-dependent recycling was absent, whereas rab11-dependent recycling predominated. Transferrin recycling via the rab4 pathway was similarly perturbed in NPFs. Compared with normal HSFs, EEs in NPFs showed high cholesterol levels and an altered organization of rab4. In vitro extraction of rab4 (but not rab11) with GDP dissociation inhibitor was severely attenuated in NPF endosomal fractions. This impairment was reversed with cholesterol depletion of isolated endosomes or with high-salt treatment of endosomes. These data suggest that abnormal membrane recycling in NPFs results from specific inhibition of rab4 function by excess cholesterol in EEs.